CN117683702A

CN117683702A - Teleosts endothelial cell line and construction method and application thereof

Info

Publication number: CN117683702A
Application number: CN202311598000.1A
Authority: CN
Inventors: 王蓓; 陈杨慧; 黎源; 蔡佳; 黄瑜
Original assignee: Guangdong Ocean University
Current assignee: Guangdong Ocean University
Priority date: 2023-11-28
Filing date: 2023-11-28
Publication date: 2024-03-12

Abstract

The invention discloses a teleosts endothelial cell line and a construction method and application thereof, belonging to the technical field of cell models. The invention adopts a pancreatin digestion mode to separate and obtain a cell line, and an astrocyte conditioned medium containing a plurality of induction factors is added in time during the culture period, so that the induced TVEC-01 cell line has the biological characteristics of more brain microvascular endothelial cells, and the preservation number is CGMCC NO.45733. The endothelial cells can pass through more than 80 passages without losing barrier functions, and the mechanism related to a plurality of biological processes of the teleosts can be researched; is used for researching in vitro relevant mechanisms of fish vascular diseases; in vitro drug screening of vascular related diseases and research of related targets; TVEC-01 cells construct an in vitro blood brain barrier model. The invention uses the model to carry out infection experiments of the streptococcus agalactiae of meningitis pathogenic bacteria, and provides a research tool for the cell-crossing path of the streptococcus agalactiae.

Description

Teleosts endothelial cell line and construction method and application thereof

Technical Field

The invention belongs to the technical field of cell models, and particularly relates to a teleosts endothelial cell line, a construction method and application thereof.

Background

A special barrier structure exists between the Central Nervous System (CNS) and the blood circulation system, called the Blood Brain Barrier (BBB), consisting mainly of endothelial cells, astrocytes, etc. The BBB has a certain ability to restrict the passage of substances and thus is an important reason for the ability of the central nervous system to stabilize and avoid infection by a variety of pathogens. In the blood brain barrier, tight Junctions (TJs) between endothelial cells are critical structures that prevent various toxins and pathogens from entering the central nervous system, and can close the intercellular spaces, which together with endothelial cells form the basis of the blood brain barrier. However, although the presence of the BBB blocks the entry of most of the pathogenic microorganisms and harmful molecules of certain pathogens into the brain, some pathogens such as streptococcus agalactiae, nervous necrosis viruses, etc. still break the blood brain barrier into the central nervous system to induce brain diseases. Once the central nervous system is developed, the drug delivery is difficult due to the existence of the blood brain barrier. In recent years, researchers have found that receptor targeting peptides, monoclonal antibodies, and the like, which can be modified by liposomes, bind to transferrin receptors, lactoferrin, and the like for targeted administration, and that this is not isolated from endocytosis of endothelial cells. In addition, endothelial cells are also indirectly involved in the regulation of physiological activities within the brain. Glucose metabolites produced by endothelial cells can enter the brain and be processed into neurotransmitters that are involved in physiological activities. More importantly, studies have shown that endothelial cells play a decisive role in blood brain barrier function, and that endothelial cells are able to limit ion movement even in the absence of astrocytes.

With the continued penetration of BBB structure research and the advent of various scientific techniques, in vitro BBB research and application has been increasingly emphasized. The preparation of the in vitro BBB with low cost, high simulation and high repeatability is particularly critical for exploring the normal physiological functions of the BBB, explaining the mechanism of BBB dysfunction under disease states, shortening the development period of CNS drugs and ensuring the curative effect of the drugs. In the field of biology, in vitro cell culture can provide an important research tool for biological research. The cell line can be used as a basic material and tool for many aspects such as virus identification, vaccine research and development, gene screening and analysis, environmental poison detection and the like. It is widely appreciated by researchers because of its low cost and good reproducibility. Although fish cell lines from different species are currently established, so far the report of establishing endothelial cell lines in teleosts has been a cursory number, and no tools have been explored as to whether they could be used as a means of constructing the extracellular blood brain barrier. This makes the construction of the blood-brain barrier in fish in vitro particularly difficult.

Disclosure of Invention

The invention aims to provide an endothelial cell line of teleosts, a construction method and application thereof, wherein the endothelial cell line has the same function as an in-vivo endothelial cell, and can be widely applied to in-vitro modeling, disease diagnosis and treatment.

The invention provides a teleosts endothelial cell line TVEC-01 with a preservation number of CGMCC NO.45733.

Preferably, the teleosts endothelial cell line TVEC-01 is derived from Nile tilapia.

The invention also provides a construction method of the teleosts endothelial cell line TVEC-01, which comprises the following steps: selecting healthy and active teleosts, obtaining cells by a pancreatin digestion method, and purifying the obtained cells for a plurality of times to obtain uniform cells in a cobblestone-like form, namely a teleosts endothelial cell line TVEC-01;

and adding a Nile tilapia astrocyte conditioned medium in the culture and passage process, wherein the Nile tilapia astrocyte conditioned medium is an L-15 medium containing 10% FBS for culturing the Nile tilapia astrocyte with good growth state.

Preferably, when the teleosts are nile tilapia, the cells are obtained by pancreatin digestion of brain tissue.

Preferably, during said culturing and passaging, purification is performed using differential adherence.

The invention also provides application of the teleosts endothelial cell line TVEC-01 in biological materials for growth, development and wound healing of teleosts.

The invention also provides application of the teleosts endothelial cell line TVEC-01 in screening medicaments for vascular related diseases, developing medicaments for vascular related diseases and/or vascular related disease related targets.

The invention also provides application of the teleosts endothelial cell line TVEC-01 in constructing an extracellular blood brain barrier model.

Preferably, the external blood brain barrier model is applied to the mechanism research of central nerve injury caused by the breakthrough of meningitis pathogenic bacteria through the blood brain barrier.

The invention also provides application of the teleost endothelial cell line TVEC-01 in constructing a model of streptococcus agalactiae infection of meningitis pathogenic bacteria.

The beneficial effects are that: the invention provides a teleosts endothelial cell line TVEC-01, which comprises the steps of obtaining cell sediment by adopting a pancreatin digestion mode, adding a nile tilapia astrocyte culture medium in time during the culture period, providing a brain microenvironment for the growth of endothelial cells, and continuously inducing the TVEC-01 cell line to have the biological characteristics of brain microvascular endothelial cells. And then continuously purifying the endothelial cells by using a differential adherence method to reduce the interference of other cells until the cell morphology presents a complete and typical 'paving stone' shape. The endothelial cell TVEC-01 not only expresses a plurality of endothelial cell marker genes, but also has a tight connection structure and the like, and is continuously transmitted to more than 80 generations, so that the barrier function is not lost. The TVEC-01 cell line can not only grow in various environments, but also carry out various experiments such as cell migration, in vitro vascular synthesis, ingestion of Low Density Lipoprotein (LDL), in vitro blood brain barrier model construction and the like, and provides research tools for the mechanism that the pathogenic bacteria of meningitis, streptococcus agalactiae breaks through the blood brain barrier to cause central nerve injury and bacteria evade host immunity.

Description of biological preservation

Nile tilapia cells TVEC-01 are preserved in China general microbiological culture Collection center (CGMCC) for 10 months and 25 days in 2023, and specific addresses are CGMCC No.45733, which is the institute of microorganisms of national academy of sciences of North Chen West Luo No. 1, chao, beijing, and the preservation number is CGMCCNO.45733

Drawings

FIG. 1 is a schematic diagram of the morphology of the teleosts endothelial cell line TVEC-01, wherein A: passage 10, B: passage 20, C: passage 30, D: passaging for 60 generations;

FIG. 2 is a graph of the identification of endothelial cell lines;

FIG. 3 is a graph showing the results of induction of TVEC-01 cell blood brain barrier properties by a culture medium conditioned by astrocytes of Nile tilapia;

FIG. 4 is a graph showing the results of optimization of culture conditions for the teleosts endothelial cell line TVEC-01, wherein A: influence of different types of culture media on cell growth under the same serum content condition; b: the influence of different serum concentrations on cell growth under the same type of culture medium and culture temperature conditions; c: influence of temperature on cell growth;

fig. 5 is a graph of cell morphology and trypan blue staining after thawing TVEC-01 by cryopreservation (scale = 100 μm), wherein red arrows indicate dead cells after cryopreservation;

FIG. 6 is a graph showing the results of a test for TVEC-01 cell migration ability;

FIG. 7 is a graph showing the results of in vitro angiogenesis experiments with TVEC-01 cells;

FIG. 8 is a graph showing the uptake results of fluorescent-labeled low density lipoprotein (Dil-LDL) by TVEC-01 cells;

FIG. 9 is a graph showing the results of the detection of TVEC-01 cell blood brain barrier properties;

FIG. 10 is a graph showing the effect of Streptococcus agalactiae on TVEC-01 cells.

Detailed Description

The teleosts endothelial cell line TVEC-01 is preferably derived from Nile tilapia, and the identification of the markers of endothelial cells from the gene level and the protein level is positive, so the teleosts endothelial cell line TVEC-01 belongs to teleosts endothelial cell lines. After continuous passage, the cell line TVEC-01 still has good growth condition and the appearance form of the cell line is typical of endothelial cells; the method can not only grow in various environments, but also carry out various experiments such as cell migration, in vitro vascular synthesis, ingestion of Low Density Lipoprotein (LDL), in vitro blood brain barrier model construction and the like, and provide research tools for the mechanism that the meningitis pathogenic bacteria streptococcus agalactiae breaks through the blood brain barrier to cause central nerve injury and the bacteria evade host immunity.

In the present invention, when the teleosts are nile tilapia, the cells are preferably obtained by subjecting brain tissue to pancreatin digestion. The present invention preferably further comprises immersing the tissue in a PBS solution containing a plurality of antibiotics, washing off surface antibiotics after immersing, mechanically crushing, such as shearing, the tissue, and then performing the pancreatin digestion. In the invention, when the pancreatin digestion is carried out, the crushed tissue is preferably placed in pancreatin solution containing EDTA for tissue digestion, 400g of the tissue is centrifuged for 3min after digestion to collect cell sediment, and the cell sediment is inoculated into L-15 culture medium (Gibco) containing 20% serum, and is cultured at constant temperature of 25 ℃ and liquid change is carried out every 3 days.

In the invention, when the liquid is changed, the mixed culture medium added with the condition culture medium of the astrocytes of the nile tilapia is preferably changed, and more preferably, the mixed culture medium comprises the following culture medium with the volume percentage: 50% fresh L-15 medium containing 20% serum with gradient reduced to 10%, 25% nile tilapia astrocyte medium and 25% TVEC-01 cell stock medium (stock solution after culturing the cells) to maintain cell growth and to induce the TVEC-01 cells to maintain more characteristics of brain microvascular endothelial cells in vivo.

The invention preferably carries out passage after the cells grow on the bottle bottom, the original culture medium is sucked away to conveniently clean the cells and digest, PBS is used for fully washing the cell surface, pancreatin is used for digesting the cells, when the cells start to shrink and round, a pipettor is used for gently blowing the cells from the bottle bottom, and then the digestion is stopped by using the L-15 culture medium containing serum. Cell pellet was collected by centrifugation and cells were thoroughly blown up with complete medium and inoculated into cell culture flasks. The cells were then observed for adherent conditions, and adherent and non-adherent cells were separated every 45 minutes and collected. With the increase of the passage times, the serum content is gradually reduced, and the cell domestication is gradually adapted to the culture medium with the serum concentration of 10% so as to save the experiment cost. After the cells grow stably and form stably, the cells are not purified through differential adherence during passage, but are passed through a ratio of 1:2, namely, the cells are divided into two parts for each passage, and are split into two cell culture flasks for half a transmission every three days.

The TVEC-01 cells have the capacity of forming blood vessels in vitro, and can provide a biological tool for the mechanism research related to a plurality of biological processes including the growth of organism organs, embryo development and wound healing of teleosts.

The TVEC-01 cells can be combined with LDL through specific targets, or LDL can enter the TVEC-01 cells through related paths, so that the TVEC-01 cells can provide biological tools for researching metabolic mechanisms of in-vivo LDL, and are beneficial to promoting in-vitro drug screening of vascular related diseases and research of related targets.

The blood brain barrier is characterized by shielding and transport effects formed by the interaction of tight junction proteins between adjacent endothelial cells. The TVEC-01 cells of the present invention express tight junction related genes (Claudin-5, occludin, ZO-1) and exhibit tight junction structures as well as express highly enriched various receptors/transporters (such as MRP, GLUT-1 and LAT) of active blood brain barrier, i.e., TVEC-01 cells can exhibit blood brain barrier properties.

The external blood brain barrier model disclosed by the invention can be preferably applied to the research on the mechanism of central nerve injury caused by the breakthrough of meningitis pathogenic bacteria on the blood brain barrier.

Experiments prove that the streptococcus agalactiae of meningitis pathogenic bacteria can invade endothelial cells of the blood brain barrier and live in the cells, can cause TVEC-01 cells to secrete a large amount of inflammatory factors, and lays a theoretical foundation for the streptococcus agalactiae to break through the blood brain barrier in a cell-crossing way.

For further explanation of the present invention, the present invention provides a teleosts endothelial cell line and its construction method and application, which are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Cell line preparation and subculture

Brain tissue of Nile tilapia was selected, and after three rinsing with PBS, a kit containing penicillin (0.1 kU/ml), streptomycin (0.1 kU/ml) and amphotericin B (0.25. Mu.g/ml) was used.

The tissue was soaked in PBS for 1min, residual antibiotics on the surface of the tissue was washed off with L-15 medium, the tissue was minced with a sterile dissecting tool and placed in pancreatin solution containing EDTA to digest the tissue for 10min. Impurities were then removed by cell-screening and digestion was stopped with serum-containing L-15 medium. After collecting cell pellets by centrifugation using a centrifugal force of 400g for 3min, cells were homogenized with an L-15 medium containing 20% serum and inoculated into a cell culture flask, and placed in a cell incubator at a constant temperature of 25 ℃. Cell growth was then observed once daily, and fluid changes were made every 3 days.

The medium was changed by: 50% of fresh serum-containing L-15 medium, 25% of centrifuged nile tilapia astrocyte medium and 25% of centrifuged TVEC-01 cell primordial medium to maintain cell growth and to induce directional TVEC-01 cells to maintain more characteristics of brain microvascular endothelial cells in vivo.

After the cells grow to the bottom of the bottle, the cells are passaged, the original culture medium is sucked away, the surfaces of the cells are thoroughly rinsed by PBS, the cells are digested by pancreatin, when the cells start to shrink and round, the cells are gently blown off from the bottom of the bottle by a pipette, and then the digestion is stopped by the complete culture medium. Cell pellet was collected by centrifugation and cells were thoroughly blown up with complete medium and inoculated into cell culture flasks.

The cells were then observed for adherent conditions, adherent and non-adherent cells were separated every 45 minutes, and non-adherent cells were collected.

With the increase of the passage times, the serum content is gradually reduced, and the cell domestication is gradually adapted to the culture medium with the serum concentration of 10% so as to save the experiment cost. After stable cell growth and stable morphology, the cells were not purified by differential adherence at passage, but passed through a 1:2 ratio, once every three and a half days.

After serial passage, the results are shown in fig. 1, the cells that were serially passed still had good growth conditions and their appearance was in the form of typical "paving stones" typical of endothelial cells.

Example 2

Identification of endothelial cell lines: identification of TVEC-01 cell endothelial markers was performed from the gene and protein level.

Total RNA was extracted from well-grown 50 th generation TVEC-01 cells with RNAiso Plus. The total RNA concentration extracted was then determined separately using a spectrophotometer. And then carrying out reverse transcription on the obtained total RNA by using a reverse transcription kit to obtain a cDNA template. NCBI was used to design endothelial cell specific gene related primers, the primer sequences are shown in Table 1. Whether the related marker gene is expressed or not is detected by PCR amplification and 1% agarose gel electrophoresis.

TABLE 1 qRT-PCR primer sequences

The cell line was further identified by immunohistochemistry. Cells were seeded into 24-well plates with slide plates, which were immunocytochemically stained three days later. After three climbs were washed with PBS, they were fixed with frozen 4% paraformaldehyde for 30 minutes. After fixation, the cells were washed three times with PBS, and incubated with the preheated antigen retrieval solution for 20 minutes. After incubation, the cells were washed three times with PBS, incubated with 0.5% Triton-100 for 20min and washed again with PBS. After 30 minutes of treatment of cells with QuickBlock blocking buffer, primary antibodies (1:200; CD34, ICAM and VE-cadherein) were added and reacted at room temperature for 3 hours, and negative control was added with PBS. After three washes in PBS, cells were left with FITC goat anti-rabbit IgG (H+L) (1:200) for 1 hour at room temperature. The secondary antibody was removed and then washed three times with PBS. Subsequently, one drop of DAPI was added dropwise for labeling nuclei, followed by observation under a fluorescence microscope.

As a result, as shown in FIG. 2, TVEC-01 cells were positive for both gene-level and protein-level identification of markers of endothelial cells, which were identified as endothelial cell lines.

Example 3

Induction of TVEC-01 cell blood brain barrier characteristics by Nile tilapia astrocyte conditioned medium to obtain Nile tilapia astrocyte (TA 02 group) and TVEC-01 with good growth stateCells (TVEC-01 group) 1X 10 per well ⁵ The cells were inoculated into 6-well plates, after cell attachment, the culture medium of TVEC-01 cells was changed to astrocyte conditioned medium (TVEC-01+AM group), and the culture medium of astrocytes was changed to endothelial conditioned medium as a corresponding control group (TA 02+ECM group). After cells per well are full, total RNA per well sample is extracted using RNAiso Plus. The total RNA concentrations extracted were determined separately using a spectrophotometer. The reverse transcription kit carries out reverse transcription on the total RNA to obtain a cDNA template.

The expression level of endothelial cell markers, tight junctions and transport related genes of each group of samples was detected by qRT-PCR, and the primer sequences are shown in Table 1. The nile tilapia beta-actin gene is used as an internal reference. Use 2 ^-ΔΔCt The relative gene expression levels of the resulting data were analyzed by the method.

As shown in FIG. 3, the expression level of the endothelial cell-related genes of the TVEC-01 and TVEC-01+AM groups is significantly higher than that of the TVEC-01 group compared with that of the TA02 group, which indicates that the astrocyte culture medium plays an important role in maintaining the endothelial cell-specific transport function, barrier function and the like of the blood brain barrier.

Example 4

Optimization of culture conditions of teleosts endothelial cell line TVEC-01 cells

Screening of the optimal culture medium of the teleosts endothelial cell line TVEC-01: l-15, DMEM, MEM and M199 medium was prepared at a serum concentration of 10%. Digestion of endothelial cells in good growth conditions was performed by using L-15, DMEM, MEM and M199 medium, respectively, at 0.2X10 cells per well ⁴ The cells were inoculated into 6-well plates and incubated in a cell incubator at 25 ℃. Afterwards, 3 wells are counted in parallel every day, sampling is carried out for 7 days continuously, and TVEC-01 cell growth curves are drawn by taking the culture days as the abscissa and the cells as the ordinate.

As a result, as shown in FIG. 4A, TVEC-01 was able to grow rapidly in the medium of L-15 and hardly in the medium of DMEM, and therefore, the optimal basal medium for TVEC-01 cell growth was L-15.

Teleosts endothelial cell line TVEC-01 blood optimumScreening of clear concentration: preparing L-15 culture medium with serum concentration of 5%, 10%, 15% and 20%, taking endothelial cells with good growth state, digesting, and respectively using L-15 culture medium with serum concentration of 5%, 10%, 15% and 20% to make cell at 0.2X10 per well ⁴ The cells were inoculated into 6-well plates and incubated in a cell incubator at 25 ℃. Afterwards, 3 wells are counted in parallel every day, sampling is carried out for 7 days continuously, and TVEC-01 cell growth curves are drawn by taking the culture days as the abscissa and the cells as the ordinate.

As a result, as shown in FIG. 4B, TVEC-01 grew fastest at 20% serum concentration, there was no significant difference in growth rate at 15% serum concentration and 10% serum concentration, and grew slower at 5% serum, so 10% serum concentration was preferred in normal cell culture.

Screening of optimal culture temperature of teleosts endothelial cell line TVEC-01: endothelial cells in good growth state were digested, and the cells were plated in a medium containing 10% FBS at a ratio of 0.2X10 per well ⁴ The cells were inoculated into 6-well plates, placed in an incubator at 18 ℃,25 ℃,32 ℃,39 ℃ respectively, then counted in 3-well replicates per day, sampled continuously for 7 days, and a TVEC-01 cell growth curve was drawn with the days of culture as abscissa and the cells as ordinate.

As a result, as shown in FIG. 4C, TVEC-01 was grown in a wide range, and survived at 18 ℃,25 ℃,32 ℃ and 39 ℃, but grown fastest at 25 ℃, and therefore, TVEC-01 cell culture temperature was set to 25 ℃.

Example 5

Cryopreservation and resuscitation of TVEC-01 cells

Cryopreservation of TVEC-01: taking TVEC-01 cells in an exponential growth phase, sucking away an original culture medium, fully rinsing the cell surface by using PBS at normal temperature, then using a trypsin solution containing EDTA to digest the cells, gently beating a cell culture bottle to promote the cells to fall off when the cells shrink and become round, and immediately stopping digestion by using an L-15 culture medium containing serum. And (3) after digestion, collecting cell sediment by using a horizontal centrifuge, re-suspending cells by using cell freezing solution, transferring the cells into a freezing tube, placing the cells into a program cooling box, and placing the cells into a liquid nitrogen tank for long-term storage after a refrigerator at-80 ℃ overnight.

Resuscitation of TVEC-01: taking out the frozen tube frozen with the TVEC-01 cells from a liquid nitrogen tank, quickly placing the frozen tube in a water bath for thawing, carefully shaking the frozen tube during thawing, quickly adding the frozen tube into a serum-containing culture medium after thawing, collecting cell sediment by centrifugation, blowing the cells uniformly by using a complete culture medium, inoculating the cells into a cell culture bottle, culturing in a cell culture box at 25 ℃, and changing the liquid every other day.

As shown in FIG. 5, the cell viability remained at 90% or higher after cryopreservation without significant change in cell morphology.

Example 6

Test of TVEC-01 cell migration Capacity

The in vitro migration ability of TVEC-01 cells was determined using a scratch assay. The cell suspension was inoculated into a 6-well plate, and after cells in each well were grown, cell scratches were made perpendicular to the plate with a gun head, the medium and scraped cell fragments were aspirated, and then the scratch was recorded with a microscope.

As a result, as shown in FIG. 6, TVEC-01 cells had a strong migration ability.

Example 7

TVEC-01 cell in vitro angiogenesis experiment

Pre-chilled base gel (Matrigel Basement Membrane Matrix) was added to the pre-chilled 24-well plate with a pre-chilled pipette to allow the gel to be spread evenly at the bottom of the well, and the gel-spread 24-well plate was then placed in a cell incubator to solidify. After the matrigel is solidified, the TVEC-01 cell suspension is inoculated into the holes fully paved with the matrigel, and a 24-pore plate is gently placed into a cell incubator to be incubated, and the angiogenesis is waited for.

As shown in FIG. 7, TVEC-01 cells have the capacity to form blood vessels in vitro, and can provide biological tools for the research of mechanisms involved in a plurality of biological processes including the growth of teleosts organism organs, embryo development and wound healing.

Example 8

Uptake of fluorescent-labeled Low Density lipoprotein (Dil-LDL) by TVEC-01 cells

And (3) inoculating TVEC-01 cells with good growth state into a 6-hole cell culture plate, adding DiI-LDL with a final concentration of 2 mug/ml into each hole after forming complete single-layer cells, incubating for a period of time, discarding the culture solution, washing for 2 times by using sterile PBS, and observing the condition of the TVEC-01 cells for taking DiI-LDL by using a fluorescence microscope.

As a result, as shown in FIG. 8, after TVEC-01 cells were incubated with DiI-LDL, red fluorescent signals appeared in the cells, and it was found that fluorescent proteins could bind to specific targets of TVEC-01 cells or enter the cells through the relevant pathways, and the DiI dye was excited by fluorescence to emit red signals. The TVEC-01 cells can provide a biological tool for researching the metabolic mechanism of in-vivo LDL, and are helpful for promoting the research of in-vitro drug screening and related targets of vascular related diseases.

Example 9

Detection of TVEC-01 cell blood brain barrier characteristics

To determine whether TVEC-01 cells express a blood brain barrier-related specific gene, TVEC-01 cells with good growth status were extracted with RNAiso Plus to obtain total RNA. The total RNA concentration extracted was then determined separately using a spectrophotometer. And then carrying out reverse transcription on the obtained total RNA by using a reverse transcription kit to obtain a cDNA template. The NCBI was used to determine whether the gene was expressed by PCR amplification and 1% agarose gel electrophoresis using specific primers related to the tightly-linked and transported genes, the primer sequences of which are shown in Table 1.

In order to further determine the barrier function of the blood brain barrier, the tight junction structure thereof was observed using an electron microscope. Collecting TVEC-01 cells with good growth state, gently collecting the cells with a cell scraper, centrifuging for 10min by 300g to collect cell precipitate, removing supernatant, and slowly adding 3% glutaraldehyde to fix the cells; after overnight fixation, the fixation solution was poured off, the samples were rinsed three times with 0.1m phosphate buffer, ph=7.0, 15min each time, and the samples were fixed with 1% osmium acid solution for 2h; dehydrating the sample with ethanol solution of gradient concentration for 15min at each concentration, and treating with absolute ethanol for 20min; then treating with pure acetone for 20min; treating the sample with a mixed solution of an embedding agent and acetone, gradually replacing a dehydrating agent in the tissue, and then treating the sample with a pure embedding agent overnight; and then slicing the sample, and observing the sample by using a transmission electron microscope after the sample is respectively dyed for 15min by a lead citrate solution and a 50% ethanol saturated solution of uranyl acetate.

To further determine the transport function of the blood brain barrier, the cell activity of the efflux transporter was detected using a drug accumulation assay. The functions of p-glycoprotein, BCRP1 and MRP1 were evaluated with Rhodamine 123, hoechst and DCFDA as specific substrates for TVEC-01 transport, respectively. After incubation, cells were lysed with 10% Triton X-100. Fluorescence intensity was detected using fluorescence excitation.

As a result, as shown in FIG. 9, TVEC-01 cells were able to exhibit blood brain barrier properties. The gel electrophoresis results demonstrated the expression of the closely linked related gene (Claudin-5, occludin, ZO-1) in TVEC-01 cells (FIG. 9 a). Tight-junctions were also found by transmission electron microscopy of TVEC-01 cells (b in FIG. 9). In addition, the blood brain barrier is highly enriched for gene expression of various receptors/transporters (such as MRP, GLUT-1 and LAT), and the result shows positive (c in FIG. 9). The activity of these transporters was further confirmed by accumulation experiments of fluorogenic substrates (d in FIG. 9).

Example 10

Streptococcus agalactiae breaks the blood brain barrier in a transcellular way and causes inflammation.

The tilapia streptococcus agalactiae ZQ0910 strain (published in the article: wang Bei. Screening for protective antigens of tilapia streptococcus agalactiae ZQ0910 strain based on whole genome sequence analysis and immunoprotection studies [ D ]. Peninsula: university of ocean, china: 2013:5-8.) and TVEC-01 cells were incubated, samples were fixed with electron microscope fixative, and samples were prepared and observed as described in example 9. The activated ZQ0910 carrying red fluorescent plasmid was resuspended in L15 medium. TVEC-01 cells were infected at a multiplicity of infection with moi=100, and after 12 hours of incubation, extracellular bacteria were killed by treatment with penicillin for 2 hours. The intracellular streptococcus agalactiae was observed periodically using a fluorescence microscope. In addition, in order to better understand the interaction between Streptococcus agalactiae and TVEC-01 cells, the relative expression levels of inflammatory factor related genes of TVEC-01 cells at different times after Streptococcus agalactiae stimulation were determined, and the primer sequences are shown in Table 1.

As a result, as shown in FIG. 10, transmission electron microscopy clearly enabled the observation of Streptococcus agalactiae in TVEC-01 cells (A in FIG. 10). It was confirmed that fish-derived streptococcus agalactiae invaded non-phagocytic cells. The streptococcus agalactiae was found to invade not only TVEC-01 cells but also to survive in the cell niches, escaping host immunity by fluorescence microscopy (B in fig. 10).

In addition, the inflammatory factor expression of TVEC-01 cells after streptococcus agalactiae stimulation was examined by qRT-PCR, and the result is shown in fig. 10C, in which the expression level of NF- κb gene was significantly increased (p < 0.05) after 24 hours of streptococcus agalactiae stimulation. Between 24 hours and 48 hours of stimulation, there was a significant up-regulation of the transcript levels of the pro-inflammatory factor genes TNF- α, IL-1β and IL-6. Notably, the relative expression levels of the anti-inflammatory factor related genes TGF- β and IL-10 were slightly up-regulated (p < 0.05) between 3 and 12 hours after stimulation. Bacterial attack on non-phagocytic cells is a process by which pathogens interact with the host. Upregulation of anti-inflammatory factors may suggest that streptococcus agalactiae utilizes certain strategies to evade host immunity. These results fully demonstrate that the meningitidis pathogen streptococcus agalactiae can invade and survive in the endothelial cells of the blood brain barrier and can cause TVEC-01 cells to secrete a large number of inflammatory factors. Establishes a theoretical basis for streptococcus agalactiae to break through the blood brain barrier in a transcellular way.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. The teleosts endothelial cell line TVEC-01 is characterized in that the preservation number is CGMCC NO.45733.

2. The teleosts endothelial cell line TVEC-01 according to claim 1, wherein the teleosts endothelial cell line TVEC-01 is derived from nile tilapia.

3. The method for constructing teleosts endothelial cell line TVEC-01 according to claim 1 or 2, comprising the steps of: selecting healthy and active teleosts, obtaining cells by a pancreatin digestion method, culturing and passaging the obtained cells, and purifying for a plurality of times to obtain uniform cells in a cobblestone-like form, namely a teleosts endothelial cell line TVEC-01;

4. A method of constructing as claimed in claim 3 wherein when the teleosts are nile tilapia, cells are obtained by pancreatin digestion of brain tissue.

5. A method of construction according to claim 3, characterized in that during said culturing and passaging, purification is performed using differential adherence.

6. Use of the teleost endothelial cell line TVEC-01 as claimed in claim 1 or 2 as biomaterial for teleost growth, development and wound healing.

7. Use of the teleosts endothelial cell line TVEC-01 according to claim 1 or 2 for screening drugs for vascular related diseases, for developing drugs for vascular related diseases and/or vascular related disease related targets.

8. Use of the teleosts endothelial cell line TVEC-01 according to claim 1 or 2 for constructing an extracellular blood brain barrier model.

9. The use according to claim 8, wherein the external blood brain barrier model is used for studying the mechanism of central nerve injury caused by the breakthrough of meningitis pathogenic bacteria through the blood brain barrier.

10. Use of the teleost endothelial cell line TVEC-01 according to claim 1 or 2 for constructing a model of streptococcus agalactiae infection by meningitis pathogenic bacteria.